Measurement of membrane bound IgD and IgM on B lymphocytes

An assay system for the determination of the membrane bound IgD (mIgD) and IgM (mIgM) on B lymphocytes was developed by the combination of two new ELISA methods with the results of flow cytometry after labeling with specific antibodies. The mIgD and mIgM of B lymphocytes were prepared by incubating mononuclear cells (MNCs) in Tween 20 containing buffer and repeated freeze/thaw cycles. Optimal results were achieved with 0.2-0.4% Tween 20 and two freezing cycles. With biotin-streptavidin amplification the sensitivity of the ELISA was 30 microU/ml for IgD and 0.5 ng/ml for IgM. In healthy persons 3.5 +/- 0.5 mU mIgD were detected on 10(6) IgD+ cells and 57.1 +/- 5.9 ng mIgM on 10(6) IgM+ cells. The mIgD/mIgM ratio was 0.065 +/- 0.005 mU/ng. The developed ELISA systems utilize only commercially available reagents and therefore provide a convenient reproducible tool for determining membrane bound IgD and IgM on B lymphocytes.